Enhanced osteoclast (OC) formation is a key mechanisms by which estrogen (E2) deficiency induces bone loss. E2 deficiency stimulates osteoclast (OC) formation by altering the phenotypic characteristics of mature stromal cells (SC). As a result, mature SC from ovariectomized (ovx) mice produce increased levels of macrophage colony-stimulating factor (M-CSF), a cytokine essential for the proliferation and differentiation of OC precursors. SC from ovx mice produce high M-CSF levels because of increased binding of the transcriptional initiator Sp-1 to a Sp site in the M-CSF promoter. We have recently discovered that the transcription factor Egr-1 inhibits Sp-1 induced M-CSF gene expression without itself binding to DNA. We have also found that Egr-1 interacts directly with Sp-1 forming a novel Egr 1/Sp- 1 complex. Ovx increases CKII-dependent Egr-1 phosphorylation, leading to decreased association of Egr-1 with Sp-1, and increased availability of unbound Sp-1, capable of transactivating the M-CSF gene. Thus, E2 deficiency, by increasing Egr-1 phosphorylation, decreases association between Egr-1 and Sp-1. These events result in increased levels of "free" Sp-1 available for binding to, and transactivation of, the M-CSF promoter. These findings are the basis for the hypothesis that Egr-1 (and factors which regulate its phosphorylation) play a critical role in the mechanism by which E2 blocks OC formation and prevents bone loss. In order to test this hypothesis, we propose 1) To determine the contribution of Egr-1 to the inhibitory effects of E2 on SC production of M-CSF and M-CSF induced osteoclastogenesis, bone resorption and bone loss in vivo using Egr-1 knock out mice. 2) To map the regions of Egr-1 and Sp-1 critical for the formation of Egr-1/Sp-1 complex using a yeast 2 hybrid system, a pull down-Western analysis and transfections of Egr-1 mutants in SC from Egr-1 deficient mice. 3). To determine the sites of Egr-1 phosphorylated by CKII in SC from ovx and E2 replete mice, and to investigate the role of Egr- 1 phosphorylation in decreasing the binding of Egr-1 to Sp-1. This will be accomplished by using mass spectrometry to map HPLC purified proteolytic fragments of Egr-1 phosphorylated by SC originating from ovx and E2 replete mice. A pull down-Western analysis and transfections of Egr-1 mutants in SC harvested from Egr-1 deficient mice will be used to determine the functional role of each Egr-1 phosphorylated residue in regulating the association of Egr-1 with Sp-1.